A. Human Urokinase
The fibrinolytic system is in a dynamic equilibrium with the coagulation system, maintaining an intact, patent vascular bed. The coagulation system deposits fibrin as a matrix serving to restore a hemostatic condition. The fibrinolytic system removes the fibrin network after the hemostatic condition is achieved. The fibrinolytic process is brought about by the proteolytic enzyme plasmin that is generated from a plasma protein precursor plasminogen. Plasminogen is converted to plasmin through activation by an activator.
Urokinase is one such activator. It and another activator, streptokinase, are currently commercially available. Both are indicated for the treatment of acute vascular diseases such as myocardial infarct, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion and other venous thromboses. Collectively, these diseases account for major health hazards and risks.
The underlying etiological basis for these diseases points to either a partial, or in severe cases, total occlusion of a blood vessel by a blood clot--thrombus or thromboembolus. Traditional anticoagulant therapy, as with heparin and coumarin, does nothing to directly enhance dissolution of thrombi or thromboemboli. Streptokinase and urokinase have enjoyed practical and effective use as thrombolytic agents. Until now, however, each has suffered from severe limitations. Neither has demonstrated a high affinity for fibrin; consequently, both activate circulating and fibrin-bound plasminogen relatively indiscriminately. The plasmin formed in circulating blood is neutralized rather quickly and lost for useful thrombolysis. Residual plasmin will degrade several clotting factor proteins, for example, fibrinogen, Factor V and Factor VIII, causing a hemorrhagic potential. In addition, streptokinase is strongly antigenic and patients with high antibody titers respond inefficiently to treatment and cannot remain on continuous treatment. Urokinase therapy is expensive, owing to its involved isolation from human urine or tissue culture, and it therefore is not generally accepted in clinical practice. Urokinase has been the subject of numerous investigations--See, for example, references 1-9. Presently available urokinase, as defined, is isolated from human urine or tissue culture, e.g. kidney cells (9A,9B).
The urokinase molecule exists is several biologically active forms--high molecular weight (ca. 54000 daltons) and low molecular weight (ca. 33000 daltons), each composed of single chain or two chain material. The low molecular weight form is derived from the high molecular weight form by enzymatic cleavage. Biologically active material contains the so-called serine protease portion linked, in active form, to a second chain via a disulfide bond. Any activity ascribed to the high molecular weight material is believed to be due to the similar presence of these two connected chains, the strategic disulfide bond and interruption in the sequence doubtless being located in the serine protease portion of the overall molecules (See FIG. 1). In any event, until the present invention, the identity, and hence function, of the ca. 21000 dalton residue was unknown and the assignment of activity to one or another of the known moieties of urokinase was not uncontrovertedly possible.
Recently, there was a report of another form of urokinase peptide having low, but specific activity (10, 10A). It was speculated that this material corresponds to native urokinase, a preform of the previously isolated active species described above, most probably consisting of a single chain.
Previous attempts to clone the requisite gene for urokinase with attendant hopes of attaining expression in a microbial host were not believed successful (11, 11A). See also (6).
It was perceived that the application of recombinant DNA and associated technologies, after all, would be a most effective way of providing the requisite large quantities of high quality, bioactive human urokinase, essentially free of other human protein, and derivatives thereof that retain functional bioactivity, thus admitting of the use of such materials clinically in the treatment of various vascular conditions or diseases.